Sam Rhine - Genetic Update Conferences - www.samrhine.com Genetics of Common Human Traits and Diseases….. Cloning and Stem Cells…..New Medical Applications Basic Genetic Stuff Review: I. CHROMOSOMES - CYTOGENETICS: A. Metaphase Spread - first observation was in 1956 - confirmed chromosome number = 46 B. Karyotype - chromosomes cut out and arranged by size - from tallest to shortest 1. Autosomes - #1 thru #22 2. Sex Chromosomes - XX - Female; XY - Male 3. Chromosome Anatomy - Chromosome #5 a. Centromere - constriction b. p-arm - short arm - placed up in the karyotype c. q-arm - long arm - placed down in the karyotype d. Telomeres - top and bottom of every chromosome e. Chromatids - double arms of the chromosome - fuse with banding f. Has #5 - Homo sapiens chromosome #5 4. Chromosome Identification - How do you tell them apart? a. Chromosome Banding - unique sets of stripes on all the chromosomes b. Permits individual chromosome identification = 'Bar Code" c. International System of stripes and numbers d. Ideogram - Idealized Diagram of banded chromosomes e. Pinpoint Break Points…..1q23 f. Pinpoint Gene Locus (Loci) = address…..Rh - 1p34; UDGP - 1q21
Transcript
Sam Rhine - Genetic Update Conferences - www.samrhine.com
Genetics of Common Human Traits and Diseases….. Cloning and Stem Cells…..New Medical Applications
Basic Genetic Stuff Review: I. CHROMOSOMES - CYTOGENETICS:
A. Metaphase Spread - first observation was in 1956 - confirmed chromosome number = 46
B. Karyotype - chromosomes cut out and arranged by size - from tallest to shortest
1. Autosomes - #1 thru #22
2. Sex Chromosomes - XX - Female; XY - Male
3. Chromosome Anatomy - Chromosome #5a. Centromere - constrictionb. p-arm - short arm - placed up in the karyotypec. q-arm - long arm - placed down in the karyotyped. Telomeres - top and bottom of every chromosomee. Chromatids - double arms of the chromosome - fuse with bandingf. Has #5 - Homo sapiens chromosome #5
4. Chromosome Identification - How do you tell them apart?a. Chromosome Banding - unique sets of stripes on all the chromosomesb. Permits individual chromosome identification = 'Bar Code"c. International System of stripes and numbersd. Ideogram - Idealized Diagram of banded chromosomese. Pinpoint Break Points…..1q23f. Pinpoint Gene Locus (Loci) = address…..Rh - 1p34; UDGP - 1q21h. High Resolution Banding - see more bands - Cystic Fibrosis - 7q31.2
C. Syndrome: a recognizable pattern of anomalies, a group of signs or symptoms, that occur together and characterize a particular abnormality
D. Chromosome Syndromes: 1 in 160 live births / 50% of a miscarriagesMCA babies (Multiple Congenital Anomalies
1. Trisomy 16 - most common chromosome syndrome - always miscarries2. Trisomy 21 - most common chromosome syndrome in live born babies
a. Down Syndrome - 1/800 - Dr John Langdon Haydon Down - 1866b. Most common GENETIC cause of Mental Disabilityc. Principle Features - Syndrome Diagnostic Criteriad. Best of the whole chromosome syndrome childrene. Karyotype - Trisomy 21f. Cause - Non-Disjunction of pair 21 on egg or sperm formationg. Monosomy 21 - other possibility with NDJ of #21
h. Parents are usually fine - error in formation of egg or spermi. 90% Maternal NDJ / Increase of DS with increasing Maternal agej. DSCR - Down Syndrome Critical Region at 21q22.1 = tripled for DS
3. Trisomy 18 - Edwards Syndrome - NDJ pair 184. Trisomy 13 - Patau Syndrome - NDJ pair 135. Monosomy X - Turner-Ulrich Syndrome - NDJ of XXs or XY6. XXY Syndrome - Klinefelter Syndrome - NDJ of XXs or XY
E. Why does a baby with an extra chromosome have a diability?
1. Why MCA?2. Why is Trisomy 21 best?3. Principle of Duplicate Balance - Genes usually come in pairs4. Trisomy 21 - 231 genes on chromosome #21 are in triplicate
a. Triple dose of all the proteins from the genes on chromosome #21b. Leukemia increaec. Fewer solid tumorsd. APP Inherited Alzheimer gene 21q21 - Down Syndrome all develop Alzheimere. DSCR-1 Gene tripled - deficient number of dendritic spines = MR
II MONOGENIC INHERITANCE - SINGLE GENE PAIR - MENDEL'S RULES of INHERITANCE
19th Century - Inheritance thought to be BLENDING - even mixture from both parents
A. Johann Gregor Mendel1. Monk - Church of the Assumption - St. Thomas Monastery in Brno, Czech Republic2. born: July 20, 1822 - died January 6, 18843. published work on peas - 1866 / forgotten / Rediscovered ~19004. "My time will come, people will see it one day, it doesn't matter that I will not be there"5. Beginning of Particulate Inheritance - inheritance via particles called genes
Pairs of genes that…..1. Segregate and 2. Independently Assort
20th Century - Inheritance understood to be PARTICULATE
B. Monogenic Reference Sites:
1. OMIM - Online Mendelian Inheritance in Man - ~20,000 with code numbers http://www.ncbi.nlm.nih.gov/sites/entrez?db=omim
2. Genes and Disease - summaries of ~80 common monogenic conditions http://www.ncbi.nlm.nih.gov/books/NBK22183/?depth=2
One Parent Affected / One Parent OK / Passed to half the children / 50%
1. Brachydactly - first recognized AD in humans - OMIM = 112500 - multiple types
2. Huntington Disease - OMIM = 143100 - 4p16.3 - Late Onset - shows up in 40's or 50's CAG Triplet Repeat Mutation - gene elongates every generationMutant HTT Huntingtin Protein - leads to loss of striatal neurons in brain
3. Neurofibromatosis - OMIM = 162200 - 17q11.2Causes small benign tumors - neurofibromas - may be mild to very severe
4. Marfan Syndrome - OMIM = 154700 - 15q1.5
Tall; slender; long arms, legs and fingers; prone to aortic aneurysm5. Alzheimer Disease - ~1% of cases inherited - APP = Amyloid Precursor Protein
APP gene > APP Protein > cleaved to become Amyloid Beta > accumulatesD. AUTOSOMAL RECESSIVE INHERITANCE - AR
Both Parents OK / Both Parents are Heterozygous Carriers / 1 in 4 / 25%
What does 1 in 4 mean - shuffle the four Aces - draw a card - Ace of hearts = aa
1. Cystic Fibrosis - OMIM = 219700 - 7131
Abnormal CFTR membrane protein - leads to abnormal cell secretionsRespiratory complications and infections - more common in Caucasians
3. Fragile X Syndrome - OMIM = 309550 - Xq27.3 - bottom of long arm of X
Moderate to Severe MR / Autism in ~50% / long face / large ears / large jaw
CGG Triplet Repeat of Mutation in FMR1 gene - at Fragile Site on X chromosome
Most common Inherited Cause of Mental Retardation
Diminished number of Dendritic spines - Therapy CTEP inhibition of mGluR5
4. Happiness Gene - sex limited - only in females - low MAOA levels X linked recessive deficiency that Lyonizes in females
Genome and Genes Review:I. INTRODUCTION: Genome / Genes / Gene Control …..September 1, 2012
A. Genome: the sum total of all the genetic information for any biologic organism
1. DNA - double strand2. RNA - single strand3. Expressed as the total number of nucleotides
a. Human Genome: ~3,000,000,000 nucleotides pairs, ~20,000 genesb. HIV Genome 9,749 nucleotides 9 genes
4. Human Genome:
~1.5% - 'Coding' DNA………carries DNA Code of 20,000 protein producing genes ~98.5% - 'Non Coding' DNA…'Junk' DNA
B. Gene:
1. 'Coding' DNA Genes - carry the genetic code to make a protein
a. sequence of DNA responsible for the production of a specific protein moleculeb. DNA double strand with promoter (on/off) region in nucleus b. DNA transcribed into mRNA (transcript) in nucleus
transcript with genetic code, plus 5' and 3' UTRs - UnTranslated Regionsc. Transcript translated into protein at the ribosome - at endoplasmic reticulumd. both Coding and Non Coding DNA segments within almost all Coding Genes
Coding Segments - Exons - part of the 1.5% Coding DNANon Coding Segments - Introns - part of the 98.5% Non Coding DNA
e. RNA Processing - occurs in the nucleus - at the SpliceosomeRemove the Introns / Splice the Exons together
2. 'Non Coding' Genes - carry a code to make RNA only
a. sequence of DNA responsible for the production of a specific RNA molecule
b. produce ~500 small non coding RNAs - microRNAs - negative gene control
3. Coding and Non Coding DNA in Coding Genes:
a. EXOME - sum total of all the Coding DNA in all the Exons - 1.5%b. INTROME - sum total of all the Non Coding DNA in all the Introns - 98.5%c. Exome plus Introme = ~30% of the genome, therefore ~70% 'Junk'd. 500 Non Coding microRNA genes in the 70%e. Most of the microRNA genes are in the 'Junk' - but some are in the introns
microRNA gene in an intron - 'Gene within a Gene' - mirTRONf. mirTRON - Non Coding DNA gene exists with an Intron of a Coding DNA geneg. Total Number of Human Genes: ~20,500…..20,000 Coding + 500 Non Coding
h. Summary - Coding DNA Genes contain both:Coding DNA (exons) and Non Coding DNA (introns)
Non Coding DNA Genes contain:Non Coding DNA only
C. GENE CONTROL = GENE REGULATION - ON / OFF MECHASNISMS
1. Transcription Factors - turn transcription ON and OFFa. attaches to the Promoterb. promoter acts as 'Docking Site'c. TF Activators - bind to promoter and turns genes ONd. TF Repressors - bind to promoter and turns gene OFFe. Enhancers - can also bind to DNA and enhance protein output
2. microRNA = miRNA - negative regulators - turn genes OFFa. microRNA, ~22 nts, complementary to and binds to 3' UTR of transcriptb. helps usher RISC (RNA Induced Silencing Complex) to the 3' UTRc. RISC blocks the ribosome > blocks translation > turns gene offd. referred to as miRNA 'Gene Silencing'e. microDNA - newly discovered type of normal human cellular DNA
small circles of non-repepitive DNA, 200-400 bp in lengthwidespread in all human somatic cellsproduced by chromosomal microdeletions
3. Epigenetics - 'Epi' means upon / on top of / above and beyond the DNA
a. Chromatin - epigenetic control mechanism acts on chromatinDNA (Genome) interacting with Histone ProteinsDNA / Histone Complex
b. Histone Proteins: H1, H2A, H2B, H3, H4NCP - Nucleosome Core Particle - octomer: 2-H2A, 2-H2B, 2-H3, 2-H4Nucleosome - DNA wrapped around the NCPNucleosome Fiber - strand of multiple nucleosomesDNA…..'Never Acts Alone'
c. Gene Contol: the Degree of Chromatin Compactioni. Methylation of Cytosine in the DNA of Chromatin at CpG Islands
add methyl groups - compact = 'Closed' chromatin = OFFlose methyl groups - loose = 'Open' chromatin = ON
ii. Acetylation of Histone Proteins = 'Open' chromatin = ONd. Methylation and Acetylation
Epigenetic Marks on the ChromatinRemodels the Chromatin Architecture
e. Enzyme Controlled Mechanism: Methylation Enzymes: DNMT - DNA Methyl Transferase
f. EpigenomeChromatin modified - DNA sequence does not change - 'above & beyond'Genome - 3,000,000,000 nucleotides of DNAEpigenome - Chromatin modifications - determines all of our phenotypesModification is an ongoing, life-long process, affected by the environment
Identical twins - two persons who are genetically identical but may not be epigenetically identical > different phenotypes
GENOME - stable - does not change
EPIGENOME - fluid - changes all the timeEpigenetic modifications can be passed through the gem line!!
D. ENCODE - ENCyclopedia Of Dna Elements - new information as of September 5, 2012
1. International Consortium
2. 32 Research Institutions
3. 442 Consortium members
4. 10 Year Genome Annotation Research Project
5. 30 Coordinated Research Papers released Sept 5, 2012
6. 4 Major Journals: Nature, Science, Genome Research and Genome Biology
7. New Genome Findings:
a. many non coding parts of the genome…..the 'Junk'
contain 'Docking Sites' where control proteins effect gene expression of both nearby and distant genes…..How Many?
2,890,000 - newly discovered Docking Sites…..in the 'Junk'
~200,000 active in any given cell at any one time
8. Gene Switches do not turn genes ON /OFF - they are 'Dimmer Switches'
9. Also in the 'Junk':
a. sncRNAs - small non coding RNAs:
20 to 200 nucleotides - microRNA,, rRNA, tRNA - ~500
b. lncRNAs - long non coding RNAs:
200 to 1000s of nucleotides - control Coding genes - ~9,500
10. 80% of the genome nucleotides now have a biochemical function!
80%.....going to be 100% soon!
11. Total number of genes in the Human Genome? ~30,000
20,000 Coding Genes + 10,000 Non Coding Genes = 30,000
Genetics of Common Human Traits and Diseases….. and their Multifactorial / Polygenic Origins
INTRODUCTION: GENOMIC MEDICINE - NEW ERA of HUMAN GENETICS
A. OLD: Genetic Medicine - the use of knowledge about single genes to improve the diagnosis and treatment of single gene diseases…..~20,000
B. NEW: Genomic Medicine - the use of knowledge about the entire GENOME
1. plus NONGENOMIC factors that effect health and disease;2. new diagnostic and therapeutic approaches to help3. understand COMMON MEDICAL conditions4. by studying POPULATIONS of people
5. Genomic Medicine - there is no 'Normal'…..No Normal human genomea. 'Variants' - millions of differences among individuals in a populationb. 'Mutation' - DNA variant that is pathologic / causes disease
"We are all Mutants"6. Person to Person Genomic Sequence - 99.6% identical DNA Sequences
0.4% different DNA Sequences a. 0.4% different = 24,000,000 nucleotides in the genomeb. 24,000,000 variants in DNA sequence between you and any other person
c. Fragile X Syndrome / Hemophilia / Duchene Muscular Dystrophy - XLRd. Retinitis Pigmentosa - AR or AD or XLR
EASY to Understand - via Punnett Square - but most conditions are very RARE
3. Why don't we study the genetics of…..Common Conditions:a. Adult Type 2 Diabetes / Macular Degeneration / Hypertensionb. High Cholesterol / Heart Disease & Stroke / Cancer & Tumorsc. BiPolar Disorder (Manic/Depressive) / ADHD - Attention Deficit / Alzheimerd. Alcohol Dependency / Autism Spectrum - ASD / Epilepsy / Asthma / etc.
…..COMMON DISEASES…..but COMPLEX GENETIC TRAITS
4. Why don't we study the genetics of …..Autoimmune Diseases:1. Type 1 Diabetes (insulin dependent) / Rheumatoid Arthritis / Multiple Sclerosis2. Lupus Erythematosis / Scleroderma / Crohn's Disease / Grave's / Psoriasis
…..COMMON DISEASES…..but COMPLEX GENETIC TRAITS
C. MULTIFACTORIAL INHERITANCE - MANY FACTORS = COMPLEX
1. Classic Genetics in the Classroom - everything in three neat boxes:a. BOX 1. Cytogenetics - Chromosomes - Karyotype - Most are very RAREb. BOX 2. Monogenic - Mendelian - Punnett Square - Most are very RAREc. BOX 3. Multifactorial - Complex Genetic Traits - COMMON DISEASES
Common Diseases - Common Medical Conditions
2. HYPOTHESIS that might Explain Complex Diseases:
a. 'COMMON DISEASES / COMMON VARIANTS HYPOTHESIS'b. Persons with the same Common Disease….. would have a unique set of DNA Variants in Commonc. Variant = DNA change found in 1% to 5% of the populationd. Quantitative Human Traits:
Continuous Distribution in a PopulationMost Common Diseases are Part of a Quantitative Trait
3. Genetic Componenta. runs through the familyb. twin study evidence - MZ v. DZc. but no Mendelian inheritance pattern - no AR, AD, or XLR
d. chromosomes and karyotype are finee. many genes involved - Polygenic - Polygenes (>500 polygenes for height)d. Environmental Factors involved…..Multifactorial!
4. HERITABILITY: H - the proportion of Phenotype variation for a particular trait that is strictly due to Genetic differences in a certain population at a certain time
a. Height H = 80% E = 20% b. Cystic Fibrosis H = 90% E = 10%c. Adult II Diabetes H = 55% E = 45%d. HIV / AIDS H = 5% E = 95%e. FASD / FAS H = 5% E = 95%f. Autism Spectrum H = 90% E = 10%
5. HUMAN QUANTATIVE TRAITS a. easily measurable traits - easy to quantifyb. continuous distribution in a populationc. in a general population - normal distribution - bell shaped curve Low / Mean / High 68% of population within one standard deviation of mean
Height Finger Print Ridge Count Blood Pressure
Blood Glucose Level IQ
6. IF we could find one of the genes for a quantitative trait
a. locate it's QTL - Quantitative Trait Locus - e.g. 6p24.2b. then check the genes at 6p24.2 from human genome mapc. relate the polygene to a function - what does that gene do?
d. how could that gene contribute to that quantitative trait
7. Flipping Pennies Model:
a. each penny represents one polygene for a quantitative trait - ten polygenes i.e. height, blood pressure, blood glucose level
b. Height: H = 80% - Heads = Tall gene Tails = Short gene
c. Polygenes - Additive or Cumulative - NOT Dominant & Recessive
d. Two average height parents usually have an average height children - most likely BUT two average height parents can have a very tall child
AND two averge height parents can have a very short child e. Medical Conditions - Threshold effect:
Blood Pressure: Most people have average blood pressure
High Blood Pressure - cross threshold - Cardiac Risk Low Blood Pressure cross opposite threshold - BP concerns
Blood Glucose Level: Most people have average blood glucose levels
High Blood Glucose - cross the threshold - DiabetesLow Blood Glucose - cross the opposite threshold -
Neuron Synapse Development:
Most people have the average number of synaptic connectionsFewer Synaptic Connections - cross threshold - Autism - ASD Extra Synaptic Connections - cross opposite threshold -
schizo
f. TWO perfectly NORMAL parents can have an AFFECTED child!!
POLYGENIC INHERITANCE…..is BLENDING!
BLENDING of individual single POLYGENES!
g. MOST COMMON DISEASES are part of a QUANTITATIVE TRAIT
D. QUANTATIVE TRAITS and POLYGENES in COMMON DISEASES:
What we do NOT know…..1. How many polygenes for a particular common disease or trait?2. Where are the polygenes located - their QTLs = address3. What do those genes do - the function of that one gene?4. Can we one day prevent the medical problems?
5. One Day…..Modify the polygenes in such a way we could …..Correct a Polygenic Disese
…..stop prople from Crossing the Threshold!
E. What would it take to find the Polygenes for Common Diseases?
1. HGP - Human Genome Project - Director: Dr. Francis Collins
Largest ever Scientific Endeavor!15 Year International Cooperative effort - 20 Countries - Mainly US + UK
Began: October 1, 1990 projected completion date: Sept. 30, 2005Actual Completion Date: April 25, 2003 - Watson / Crick AnniversaryHow many human genes? 100,000 > 20,000 coding genes
Plus the addresses of all the gene loci ~3,000,000,000 nucleotide pairs
~1.5% Coding / ~98.5% Non-Coding
'Rough Draft' - June 26, 2000 - US / British joint announcement1600 Pennsylvania Avenue in DC / 10 Downing Street in London
Human DNA - we are all 99.6% Identical DNA SequenceSame among and between racial groupsNo DNA basis for the term 'Race'
Published: Nature - February 15, 2001 / Science - February 16, 2001
"To determine our DNA Sequence is to achieve an historic step forward in Human Knowledge"
2. SNPs and CNPs
a. SNPs - Single Nucleotide Polymorphisms = SNVs - Single Nucleotide Variant "snips" - found in >1% of the population - common How many SNPs in me or you? ~3,750,000
b. CNPs - Copy Number Polymorphisms = CNVs - Copy Number Variants "cnips" - found in >1% of the population - common
c. If we are all 99.6% identical in our DNA sequences
d. Differences that cause common diseases - must be in 0.4% where we are different
SNPs = SNVs - Small Scale variants - ~80% of the 0.4%CNPs = CNVs - Large Scale variants - ~20% of the 0.4%
Mutation - Rare change - associated with pathology / diseaseSNPs - Common - normal variant in the population
e. How do we find the SNPs? Search for groups of SNPs - Haplotypes
Haplotype - Sets of nearby SNPs - located close together on a chromosome HAPMAP - Haplotype Map - find the addresses of the SNP loci
Hapmap I 2005 - 1,000,000 most common SNPsHapmap II 2006 - 10,000,000 SNPsHapmap III 2010 - 1,440,616 SNPs for detailed studies
Average Person: 1 SNP every ~800 bp = ~3,750,000
f. HYPOTHESIS: COMMON DISEASES / COMMON VARIANTS
COMMON DISEASES / COMMON SNPs
F. GWAS - GenomeWide Association Studies - New England Journal Med July 8, 2010
1. Evaluate the entire genome of thousands of people
2. See if any SNPs might be Associated with a particular Common Disease
3. GWAS Example: T2D - Type 2 Diabetes - usually adult onset
a. Experimental Group: 2500 people - medically confirmed DO have T2Db. Control Group: 2500 persons - medically confirmed - DO NOT not have T2Dc. Scan their 5,000 genomes and check to see which SNPs are presentd. Question? Do the 2500 persons with T2D have a unique set of SNPs
in Common that persons without T2D DO NOT HAVE? YESe. AND…..we know the QTLs of those unique SNPS…..
one unique T2D SNP is located at 10p12.3one of the polygenes for T2D must be located at 10p12.3 - Candidate Gene
f. go to Human Genome Map - to 10p12.3 - see which genes are thereg. attempt to link that SNP to a known gene Function
4. We have TESTED the HYPOTHESIS: COMMON DISEASE / COMMON VARIANTS HYPOTHESIS
5. All GWAS results available - One Web Site: www.genome.gov/gwastudies/
a. Over 1400 hundred entries - as of June 1, 2012b. > 1600 GenomeWide Association Studiesc. > 250 Distinct Human Traits and Diseasesd. > 7291 SNP - Trait Associations - as of September 12, 2012
G. How do the GWAS tests work? Using CHIPS to find the SNPsd
1. CGH Technology - Comparative Genomic Hybridizationa. Microarray Chip = SNP Chip = DNA Chipb. Glass chip with array of DNA fragments synthesized in a laboratoryc. Fragments on chip - one side of the DNA molecule - 5' to 3' side = PROBE d. Location and sequence of all PROBE fragments on array is knowne. Each PROBE fragment on the Chip carries a known SNP sequence
Unknown DNA to be tested is chopped into fragments - 3' to 5' sideUnknown fragments are tagged with a fluorescent colored labelUnknown DNA fragments - 3' to 5' - placed on the chip with the PROBESUnknown fragments hybridize (form double helix) with known fragment
f. Laser Scanner 'reads' the fluorescent colored spots on the Chipg. Presence & Intensity of colored spots identifies the SNPs in unknown DNA
6. Blood Lipids - major indicator of heart disease 100,000 persons tested - 95 distinct gene variants 59 new - never found before
7. SNPs and I.Q. - g = general cognitive ability - Scientific American - Oct 2008
8. SNPs and Human Personality Traits: Neuroticism / Extraversion / Openness / Agreeableness / Conscientiousness / Music Ability All human behaviors polygenic??
9. SNPs and Human Facial Appearance - VisiGen - International Visible Trait Geneticsa. 5 genes found that affect human facial shapeb. 24 genes for eye and hair color - Forensic Applications - facial ID from bloodc. Hope to identify victims / perps face from DNA in blood samples at crime scenes
11. GWAS - Big Questions…..a. ONLY ~12% of GWAS SNPs are located in coding gene regionsb. ~40% of GWAS SNPs are in non-coding intronsc. ~49% of GWAS SNPs are in intergenic regionsd. may indicate intronic or intergenic gene control elements
Now we know - e. "Missing Heritability"
T2D: 18 SNPS - 18 Polygenes - H = 55% - only ~6% of H - ~94% missing
Height: 180 SNPs - only 20% of H - 80% missingLook Deeper - 500,000 persons with SNP evaluations?
f. 'Revolution Postponed' - Scientific American - October 2010I. CNVs / CNPs - Copy Number Variants = Copy Number Polymorphisms
1. Normal - 2 copies / CNP = 1 copy, 3 copies, etc.2. Large Scale Variation - ~20% of the 0.4%3. INDELS: INsertions / DELetions or Rearrangements
a. MAY be INHERITED…..passed from parents orb. MAY arise DE NOVO - mistake in gametogenesis - not passed from parentsc. Copy Number Variant - five instead of twod. Most Common Example - Down Syndrome
all genes on chromosome #21 - x34. Epilepsy / HIV / Heart / Schizophrenia / Tourette's / Obesity / Autism
J. AUTISM - Autism Spectrum Disorders - National Autism Association http://nationalautismassociation.org
1 in 88 children.....1 in 54 males.....Males : Females = 4 : 1 Neurodevelopmental disorder characterized by: 1. Social Impairments 2. Cognitive Impairments 3. Communications Difficulties 4. Repetitive Behaviors Can range from very mild to very severe. Can occur in any ethnic, socioeconomic and age groups. Some children with autism appear normal before age 1 or 2 then suddenly 'regress' and lose language
skills they had previously gained. This is the regressive type of autism. SIGNS of AUTISM: 1. No big smiles or other warm, joyful expressions by six months or thereafter. 2. No back and forth sharing of sounds, smiles or other facial expressions by nine months or thereafter. 3. No babbling by 12 months. 4. No gesturing (pointing, waving bye-bye) by twelve months. 5. No words by 16 months. 6. No two-word meaningful phrases (without imitation or repeating) by 24 months. 7. Any loss of speech or babbling or social skills at any age. EARLY SIGNS OF AUTISM: 1. Doesn't make eye contact (e.g. look at you when being fed). 2. Doesn't smile when smiled at. 3. Doesn't respond to his or her name or to the sound of a familiar voice. 4. Doesn't follow objects visually. 5. Doesn't point or wave goodbye or use other gestures to communicate. 6. Doesn't follow the gesture when you point things out. 7. Doesn't make noises to get your attention. 8. Doesn't initiate or respond to cuddling. 9. Doesn't imitate your movements and facial expressions 10. Doesn't reach out to be picked up. 11. Doesn't play with other people or share interest and enjoyment. 12. Doesn't ask for help or make other basic requests.
1. Children with autism carry a higher load of rare CNVs2. Some inherited from parents as polygenes - some having arisen de novo3. de novo CNVs account for 5-8% of simplex cases - paternal age effect4. Obesity & Underweight / Macrocephaly & Microcephaly / Autism & Schizophrenia
Examples of CNVs and Reciprocal Variants - Phenotypic Opposites
L. All GWAS results available - One Web Site: www.genome.gov/gwastudies/M. Early Genetics…..1800's-1900's - BLENDING Inheritance
Blending of Characters of Father & Mother Beginning in 1900's - PARTICULATE Inheritance - particles called genes - Mendelian
Today - BLENDING of POLYGENES from MOTHER and FATHER
N. SAGE - Study of Addiction Genetics and Environment
1. COGA - Collaborative Study of Genetics of Alcoholism2. FSCD - Family Study of Cocaine Dependence3. COGEND - Collaborative Genetic Study of Nicotine Dependence
O. TCGA - The CANCER GENOME ATLAS
1. Prostate Cancer - CaP - Prostate Cancer - 45 SNPs - predisposition SNPs2. Breast Cancer - 25 predisposition SNPs - Monthly self evaluations!
3. Colorectal Cancer - 20 predisposing SNPs4. Lung Cancer - Tumor cells contain up to 50,000 SNPs…..Cancer Causing Mutations!
P. HUMAN MICROBIOME PROJECT
1. 800,000,000,000,000 - cells in the human body a. 80,000,000,000,000 - human cells
b. 720,000,000,000,000 - Microbes: Bacteria, Fungi, Protozoac. Major Environmental Factors - Multifactorial
Q. YOUR PERSONAL TOTAL GENOME EVALUATION
1. 'The Language of Life' - book by Dr. Francis Collins - Director of NIH2. Construct Your Own Detailed Family History / Pedigree Online via HHS
www.familyhistory.hhs.gov/3. Today - Check you Total Genome - 3,000,000,000 bp - $25,0004. Partial Genome & Common SNPs - $1,0005. Faster and Cheaper Sequencing in the Future…..Whole Genome - 2014 - $1000
R. THE '1000 GENOME PROJECT' - began in January 2008
1. Collaboration among US, UK, China, Germany 2. Produce an extensive catalogue of human genetic variation - SNPs & CNPs3. Goal is to provide a resource of almost all human variants4. Will allow more extensive GWAS studies / Hapmap III is a part of it5. Over 1000 genomes of unidentified individuals from around the world6. Form the basis for ethnic group comparisons and tracking people groups7. Three Pilot Studies…..
a. Deep sequencing of Mother-Father-Adult child triosb. Light sequencing to see how data can be combined across samplesc. Cataloguing Coding DNA Exome regions of the 1000 genome Web Site: http://www.1000genomes.org/page.php
8. We each receive ~60 new mutations in our genome from our parents
S. YOUR PERSONAL DNA CHIP
1. 'Genomic Era of Medicine' - 'Personalized Medicine'2. Pharmacogenetics - Personalized Prescriptions - Personalized Cancer Therapy3. GPP - your personal Genetic Predisposition Profile - your Medical Future
a. Do you want to know?
b. Who else should know?
c. Who pays? Insurance?
d. Who else should have access?
e. Where will you keep the information?
f. Who will explain it?
4. GINA - Genetic Information Nondiscrimination Act
5. Prenatal Diagnosis and SNP evaluation of the unborn?
Your unborn baby is:
a. XY - boyb. height SNPS - he will be about 6'1"c. T2D SNPs - ~4.1% chance for adult onset Type 2 Diabetes d. ~1.9% chance of developing rheumatoid arthritise. ~2.6% chance of developing osteoporosisf. ~8.1% chance of attention deficit hyperactivity disorderg. ~4.9% chance of developing hypertensionh. ~0.6% chance of becoming alcohol dependenti. ~16.2% chance of developing prostate cancer as an adultj. ~57.7% chance of being gifted musicallyk. ~5.9% chance of having an agreeable personalityl. ~39.4% chance of being very conscientious
What if there was ~75% chance of migraines?
What if there was ~80% chance of being autistic?
First Human Prenatal Genome Evaluation reported in Spring 2012
…..its not coming - it is here!
T. SNP WHOLE GENOME EVALUATIONS - will be routine soon:
…..the 'Greatest Tidal Wave' of new medical information in world history
Cloning and Stem Cells…..New Medical Applications
I. INTRODUCTION
A. Public perception of cloningB. What is your personal stance on…..Cloning? …..Stem Cells?C. Scientific Literacy…..Be Well Informed…..Make Informed DecisionsD. Cloning: two procedures - 1. Reproductive Cloning
2. Therapeutic Cloning 3. How does it work? How are they similar? different? end point?
E. Four Main Types of Stem Cells: 1. Embryonic Stem Cells (ESCs) - first grown in lab - 1998 2. Adult Stem Cells (ASCs) - first recognized - 1961 3. induced Pluripotent Stem Cells (iPSCs) - man-made - 2007 4. Cancer Stem Cells (CSCs) - first recognized - 1997
F. Controversial - Bioethical Issues: ethical, moral, religious, political issues
II. BIOLOGIC BASIS of CLONING and STEM CELL TECHNOLOGIES:
A. Human Embryology - formation of the fetus = Applied Embryology
B. Review of Early Human Embryology1. Fetus at 49 days - size of a quarter2. Fetus at 56 days - size of a silver dollar - 220 somatic cell types 3. HOW?
C. Differentiation:1. amazing process where all 220 fetal somatic cells become 'Specialized'…..220 Ways2. "Other than making everything…..differentiation doesn't do anything!"3. 220 One Way Streets = 220 Sets of Signals4. Terminal Differentiation - end of the 220 Streets - 220 Specialized Somatic Cells5. One Way Street - Basic Tenet of Human Embryology6. What are the signals? Molecular Signal Proteins = Transcription Factors
Activate or Repress dozens to hundreds of genes at one timeDifferentiation Pathway for Pancreatic Beta (insulin producing) cells: 10 signals
7. Human Embryome Project - International effort to find all 220 sets of signals
D. Fertilization to Stem Cells…..1. Fertilization or Conception - egg and sperm unite2. Zygote = fertilized egg - size = point of a straight pin3. 2 cell embryo at ~36 hours4. 4 cell embryo at ~42 hours5. 8 cell embryo at ~48 hours > 16 cells > 32 cells > 64 cells can mix and match blastomeres to make chimeric monkeys - 6 embryos > 12 parents 6. Blastocyst at ~ 6 days
a. 'basketball' - size of the point of a pin = implantation stageb. inside blastocyst - fluid filled - one small cluster of cells = ICMc. ICM - Inner Cell Mass = Embryonic Stem Cells - ESCs - becomes fetus/amniond. 'Ten Day Rule' - ESCs only exist for 10 days under natural conditionse. ESCs first grown successfully in a lab - James Thomson - U. Wisconsin -1998
E. Embryonic Stem Cells…..
1. Why are they 'stem' - term borrowed from the plants - gives rise to all the branches2. from the ESC stem immediately come four major branches
3. these four major branches: beginnings of Adult Stem Cellsa. 220 branchesb. 220 one way streetsc. 220 sets of signalsd. 220 unique sets of epigenetic remodeling marks
4. Stem Cells do not come from the Brain Stem! Common politician misunderstanding!
F. Adult Stem Cells:
1. initial function - differentiate into 220 somatic cells in the developing fetus2. continued function - produce replacement cells the rest of your life3. Where are they today?
a. HSCs - in bone marrow (and in umbilical cord) i. producing replacement blood cells ii. i.e. produce ~15,000,000 RBC per second to replace those lost
\ b. MSCs - in bone marrow, adipose, amniotic fluid derived from pericytes i. produce new muscle, bone, cartilage, tendon, heart, vessels ii. produce paracrine signals for tissue repair, act as cellular pharmacy
c. ESCs - in lining of stomach and lining of intestine - making replacement cells
d. NSCs - in hippocampus and sub-ventricular zones of the brain
e. HSCs and MSCs are easily obtained from bone marrow or adipose used therapeutically for over 50 years - 1st BM transplant was 1959
f. Differentiation begins at around 10 days…..continues forever
g. Adult Stem Cells - term might be misleading i. ESCs - only in the early blastocyst embryo - gone by 10 days ii. ASCs - in early embryo, fetus, newborn, toddler, child, teen, adult
h. Ectoderm = NSCs, Endoderm = ESCs, Mesoderm = MSC sans HSCs
G. ESCs - Embryonic Stem Cells - unique characteristics:1. Self Renewal - Biologic Immortality / Telomerase2. Differentiate - produce any of 220 specialized cells3. Migrate - throughout the developing embryo as the fetus forms4. DNA Repair - fix potentially lethal DNA damage from radiation or chemotherapy
H. Important 'Potent' Terms that describe stem cells1. Totipotent - cell with the Total Potential to form a fetus, placenta and membranes
a. zygoteb. blastomeres up through the 8 cell embryo
2. Pluripotent - stem cell with the ability to differentiate into all 220 cell typesa. ESCs
b. iPSCs3. Multipotent - stem cells that can form many, but not all, of the 220 cells - limited
a. HSCs, b. MSCs, c. ESCs, d. NSCsIII. REPRODUCTIVE CLONING
A. Clone - a genetically identically copy of…..anything you choose
B. Robert Briggs - first to clone an animal - frogs in 1952 - 'Father of Cloning' - IU
C. Definition - reproduce an individual…..create a genetically identical copy of a particular individual…..they will be born as a new baby
D. Epigenetics - mechanism whereby chromatin…..our DNA and histones are modified in such a way that two individuals with identical DNA, might have
different phenotypes
E. First Mammalian Clone: Animal - Sheep / Name - Megan & Morag - from embryo nucleus
F. DOLLY - first clone from an adult somatic donor nucleus
G. Reproductive Cloning Procedure: Somatic Cell Nuclear Transfer - SNCT
1. Oocyte Retrieval - obtain egg from the sheep ovary2. Enucleation - remove the nucleus from the egg - surgery on the egg to remove nucleus3. SCNT - transfer donor somatic nucleus into the egg - new set of instructions the DONOR will be CLONED4. Artificial Activation of the egg > 2cell, 4 cell, 8 cell…..5. ET - Embryo Transfer from the petri dish into the uterus of a surrogate mother
H. Dolly born July 5, 1996 / dies February 14, 2003 - death from virus infection - not cloning1. Ian Wilmut's lab in Roslin, Scotland2. Dolly today: Royal Museum in Edinburgh
I. End Point for Reproductive Cloning - BABY born to surrogate mother - too risky for humans
J. Mammalian clones: Sheep, Mouse, Cattle, Pig, Goat, Gaur & Mouflon (endangered), Rabbit, Cat, Mule, Rat, African Wildcat, Dog, Water Buffalo, Horse, Ferret, Wolf, Banteng, Camel, 'Miracle' Pig in China, Wooly Mammoth?
K. Prometea - first cloned horse - donor was also the surrogate - .12% success rate Dolly - success rate - .36% - very inefficient process - why…..Imprints/Epigenetics
L. Most important lesson from Dolly:
1. a highly specialized, terminally differentiated, somatic donor nucleus, when placed in an enucleated egg, is reprogrammed back to a pluripotent state2. What is it in the oocyte that reprograms the nucleus back to pluripotency?3. Must be a cytoplasmic factor - because the egg nucleus in gone4. Guess…..SIGNALS in the cytoplasm5. How long would it take to find those signals?6. How many signals would it take to reprogram nucleus back to pluripotency?
IV. THERAPEUTIC CLONING
A. Therapeutic Cloning Procedure: SCNT1. Human Oocyte Retrieval - very difficult procedure2. Enucleation of the egg - surgery on the human egg to remove nucleus3. SCNT - transfer donor somatic nucleus to the egg - new set of instructions4. Artificial Activation of the egg > 2 cell, 4 cell, 8 cell…..5. Embryo continues to grow in lab for 6 days - clone in the petri dish
Clone of my friends embryoRemove ECSs from the embryo and grow the cells in the labEstablish a cell line from ESCs, let them grow until you have 10,000,000,000 ESCsDirected Differentiation:
a. give those cells the signals for Spinal Motor Neurons (SMNs)b. result: > 10,000,000,000 SMNs - genetically identical to my friendc. the 'DREAM' - use those cells to repair his spinal cord - he walks again!
Definition: Therapeutic Cloning = Research CloningCreate a Genetic Clone (copy) of an individual's embryo;grow ECSs from the embryo; use the appropriate signals;produce any of 22 human somatic cells to use for medical therapy
END POINT: a CELL that can be used for medical therapy
B. Applications:
1. Cell Replacement Therapy - autologous - no rejectiona. Spinal Motor Neurons - spinal cord injuryb. Pancreatic Beta Cells to produce insulin - Type I Diabetesc. Dopamine producing neurons - Parkinson Diseased. Red Blood Cells - Sickle Cell Anemiae. 216 other possibilities
Directed Differentiation of iPS > spinal motor neurons > pancreatic beta cells > dopamine producing neurons
No Rejection - No Embryos are needed
6. Tests for Pluripotency:a. Cell Morphology under the microscopeb. Cell Biomarkers for pluripotencyc. Cell induces teratoma in SCID moused. Cell with GFP followed in chimera embryos > egg and sperme. Cells form an embryo in 'Tetraploid Complementation' Test
the Definitive Test - Nature, September 3, 2009
7. Ian Wilmut - lead team that produced Dolly - full time iPS in November 2007
Special New "iPS Facilites" labs at Harvard, Toronto, Kyoto, et.al.
VI. iPSCs Applications:
1. Correct Human sickle cell in mice w/ iPS + genetic engineering - Dec 2007
2. Fibroblast iPS for Parkinson's in rat model - GFP cell sorting to stop tumors PNAS (OL) April 7, 2008
3. iPS from ALS patients (Lou Gerhig's) to study in vitro - July 31, 2008Science OL
4. iPS for: Huntington Dx, Parkinson Dx, Muscular Dystrophies, Type 1 Diabetes, ADA Deficient SCID, Gaucher Dx III, Shwachman-Bodian-Diamond Syn, Lesch-Nyhan Carrier, Down Syndrome - study in vitro - August 6, 2008
5. 25,000,000 World Wide - visually impaired
AMD - Age Related Mauclar Dystrophies Dry Form - Photoreceptor Loss / Wet Form - Neovascularization Stargardt's - Photoreceptor loss - pediatric
a. huESCs from donated blastocysts for Dry MD and Stargardt'srisk of rejection but Retina is Immunopriveleged SiteESC > Retina Pigment Epithelium - 99% pureFirst Successful Human Trials - The Lancet - January 23, 2912
b. RP - Retinitis Pigmentosa - Most Common Inherited Vision Loss AD, AR, XLR
iPS cells become Retinal Rod Cells Nature, May 3, 2012
c. Retinal Prosthesis with high resolution stimulator in sync with the retina's neural code
d. Leber Congenital Amaurosis Type 2 - LCA2
one injection of 'RPE65' gene therapy via AAV vectorrestored vision - Lancet, Nov 7, 2009
6. Hearing Loss - restore auditory spiral neurons
7. Huntington Disease - AD - CAG Triplet Repeat Mutation
a. Genetic Correction of HS Phenotypes in iPSCsHD Patient Fibroblasts > HD iPSCsHD iPSCs Genetically Corrected via Homologous Recombination
CAG72 > CAG21 Corrrected HD iPSCs > Striatum Neurons
b. patient specific, genetically corrected, HD patient iPSCs critical step for the eventual use of these cells in Replacement therapy CURE Huntington Disease?
c. turn off the mutant gene with ss-siRNA Gene Silencing delivered to ventricles
8. Mature Pulmonary Epithelium
a. Direct differentiation of iPSCs to mature airway epithelia with CFTRb. Potential therapy for Cystic Fibrosis?
9. Also iPSCs for:
a. Alpha-1-Antitrypsin Deficiency - genetically corrected iPSCs > liverb. Hutchison Guilford Progeriac. Friedreich's Ataxiad. Long Q T Syndrome - heart conditione. Fragile X Syndromef. Rett Syndrome - Autism Spectrum Model
g. Parkinson Diseaseh. Schizophrenia
10. Other Applications:
a. huiPSCs > Neural Stem Cells > Rats with Parkinson - improvementb. ALS iPSCs - Astrocytes destroying motor neuronsc. huHSCs > Red Blood Cells in vitrod. hu DiPSCsd. Spermatids from skin or ESCS
11. Direct Reprogramming: fibroblast directly to desired somatic cell - no iPSCs
a. Neuronsb. Dopamine Neuronsc. Intestinal Tissued. Alzheimer Neuronse. Hepatocytesf. Sperm
12. Man-Made Organs and Applications:
a. Teeth
b. Optic Cup Retina
c. Primitive Cerebral Cortex
d. Primitive Hypothalamus with hormone production
e. Primitive Cerebellum
f. MSCs on man-made scaffold - heart vein
e. Repair tracheal damage from tuberculosis with adult MSCs
f. Trachea made from scratch - MSCs plus man-made scaffold
g. Cartilage made from scratch - MSCs in seeded on polymer scaffold
h. Cardiac Fibroblasts make new Cardiomyocytes
i. Prenatal Diagnosis - heart valve anomaly - AF MSCs new valve
j. OI corrected before birth with maternal MSCs via cordocentesis
k. Finger Regeneration - cellular matrix powder from a pig bladder
l. Meat Lab - make burger and sausage from muscle stem cells
m. Made rat pancreas in a mouse embryo…..Human Pancreas??
o. Yamanaka - iPS Stock Cell Project in JapanHLA screening ~64,000 umbilical cord blood samplesto find 75 Donors for >80% of Japanese populationwill make iPS available for most of the Japanese population
Here are some College and Career 'Contribution' suggestions:
1. Go to you favorite Undergraduate college and obtain your Bachelor's degree. Major in biology, biochemistry, molecular biology, bio-engineering etc. Make sure you satisfy the Pre-Med requirements so you can apply to medical school if you decide that is the best route for you.
2. Medical School is four years and the curriculum is very similar at all medical schools in the US. The reason for that is that everyone must pass the same national exam after finishing medical school - therefore the schools must cover the subjects. If you pass that exam the summer after finishing medical school then you can put M.D. behind your name.
3. Residency is then 4 - 8 years of specialty training to become a pediatrician, obstetrician, orthopedic surgeon, oncologist, neurosurgeon or whatever specialty you choose. If you want to pursue a career in Tissue Engineering then you might want to get a residency with Dr. Anthony Atalla at Wake Forest University. If you want to use antibodies to stop leukemia caused by cancer stem cells you might want to do your residency in oncology at Stanford University. Keep your ‘antennae out’ during the four years of medical school - determine who is doing what you want to pursue for a career - and go do your residency with that person - he or she.
2. For those who are not interested in medical school - they might want to pursue a career in research and they will go on after their undergrad work and get their Masters and Ph.D. which may be 4 to 6 more years.
3. The Ph.D. is usually followed by Post Doctoral studies for 2 - 4 years to gain special expertise for the research career you want to follow. Then you will be ready to job on the faculty at a university to do research and teach. Others will opt to get a job doing research in industry for biotech companies. Also, some of these people are getting their Ph.D.s in biostatistics or computer science where they will help with the planning and evaluation of research data being generated.
Many major Medical Schools offer a combined M.D. / Ph.D. for a person who may one day be the chairperson of the Department of Molecular Medicine at some medical college
Another option for some will be to get a Masters Degree in Genetic Counseling. There are almost 30 places in the US where those programs are available. For more information - check out this web site:
Also Remember…..many people who will make a major contribution to all these careers in the future will do so by majoring in Education in college and will be preparing young people in the future…..as your Teachers have been preparing you!!
